The objective of this work is to identify and determine the mode of action of a humoral factor that replaces helper T cells in the induction of antibody synthesis. Spleen cells have been stimulated with Concanavalin A (Con A) under conditions which lead to the production of TRF in the culture supernatants from single T cells. These supernatants have been assayed in multiple microcultures of B cells containing two heterologous erthrocyte antigens. When produced in conditions of limiting dilution, the TRF show a segregation of biologic activities as revealed by an ability to stimulate immune responses to one erythrocyte but not another. These results indicate that TRF produced in Con A-treated spleen cultures have antigenic specificity. Activation of T cells with antigen in mice results in an increase in the frequency of antigen-specific supernatants after Con A treatment of cells in culture. We postulate these culture supernatants derived from Con A-Activated spleen cells contain receptors derived from helper T cells, and that all T cell-replacing activity observed is due to the expression of the biologic activity of these receptors. Con A-treated spleen cells produce a polyclonal mixture of T cell receptors, hence the culture supernatants are apparently antigen non-specific. In the limiting dilution analysis, which restricts the number of factor-secreting cells, antigenic specificity for erythrocyte antigens is revealed. These data support the concept that the helper effect is due to the expression of T cell receptors bound to an accessory cell type, present in athymic (nude) spleen cultures. The biologic activity has been concentrated by salt precipitation and purified by gel filtration, ion exchange chromatography and isoelectric focusing. At each step, the recovery of biologic activity is determined. The activity is found in protein of 30-40000 daltons size as estimated by gel filtration, but shows considerable heterogeneity in isoelectric properties. The activity is active at concentrations of less than 10 to the minus 10th power M, is strictly antigen-dependent in its mode of action, and exhibits no mitogenic or polyclonal properties.